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MePMe-seq: antibody-free simultaneous m6A and m5C mapping in mRNA by metabolic propargyl labeling and sequencing
Katja Hartstock1, Nadine A Kueck1, Petr Spacek1
1Institute of Biochemistry, Faculty of Chemistry and Pharmacy, University of Münster, Corrensstraße 36, 48149, Münster, Germany.
Abstract:
Internal modifications of mRNA have emerged as widespread and versatile regulatory mechanism to control gene expression at the post-transcriptional level. Most of these modifications are methyl groups, making S-adenosyl-L-methionine (SAM) a central metabolic hub. Here we show that metabolic labeling with a clickable metabolic precursor of SAM, propargyl-selenohomocysteine (PSH), enables detection and identification of various methylation sites. Propargylated A, C, and G nucleosides form at detectable amounts via intracellular generation of the corresponding SAM analogue. Integration into next generation sequencing enables mapping of N6-methyladenosine (m6A) and 5-methylcytidine (m5C) sites in mRNA with single nucleotide precision (MePMe-seq). Analysis of the termination profiles can be used to distinguish m6A from 2'-O-methyladenosine (Am) and N1-methyladenosine (m1A) sites. MePMe-seq overcomes the problems of antibodies for enrichment and sequence-motifs for evaluation, which was limiting previous methodologies. Metabolic labeling via clickable SAM facilitates the joint evaluation of methylation sites in RNA and potentially DNA and proteins.
Insights
Researchers developed a new method using clickable SAM precursors to detect and map mRNA methylation sites, including N6-methyladenosine (m6A) and 5-methylcytidine (m5C), with single-nucleotide precision.
Area of Science:
- Molecular Biology
- Epigenetics
- Biochemistry
Background:
- Internal modifications of mRNA, primarily methylation, regulate gene expression post-transcriptionally.
- S-adenosyl-L-methionine (SAM) is a key metabolic hub for these methylation processes.
Purpose of the Study:
- To develop a novel method for detecting and identifying various mRNA methylation sites.
- To enable precise mapping of methylation sites using next-generation sequencing.
Main Methods:
- Metabolic labeling using propargyl-selenohomocysteine (PSH), a clickable SAM precursor.
- Intracellular generation of a SAM analogue leading to propargylated nucleosides.
- Integration with next-generation sequencing for MePMe-seq to map N6-methyladenosine (m6A) and 5-methylcytidine (m5C) sites.
Main Results:
- Successful detection and identification of various mRNA methylation sites.
- Single-nucleotide precision mapping of m6A and m5C sites in mRNA.
- Distinguishing m6A from A_m and m1A sites using termination profile analysis.
Conclusions:
- MePMe-seq overcomes limitations of previous antibody-based and sequence-motif methods.
- Clickable SAM-based metabolic labeling facilitates joint evaluation of methylation across RNA, DNA, and proteins.
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